Enteric compounds and complexes

ABSTRACT

A complex of a therapeutic agent is disclosed in which the therapeutic agent is complexed to an ammonium ion selected from the group consisting of ##STR1## where R 1  and R 2  are the same or different and are alkyl or hydroxy substituted alkyl containing 1 to 6 carbon atoms; and 
     R, R&#39; and R&#34; are the same or different and are saturated or unsaturated aliphatics containing at least 10 carbon atoms, ##STR2## where R 1 , R 2  and R&#34; are as defined above, ##STR3## where R 3  is independently alkyl or hydroxy substituted alkyl containing at least 10 carbon atoms; and R 1 , R 2 , R 3  and R and R&#39; have the meanings given above; and ##STR4## where R 1 , R 2  and R 3  have the meanings given above. The therapeutic agent is heparin, a biologically active peptide or protein or an antineoplastic drug. The therapeutic agent may also be covalently bonded to a triglyceride type backbone to form a compound. The compound thus formed has a structural formula selected from the group consisting of ##STR5## where R and R&#39; are the same or different and are saturated or unsaturated aliphatic containing at least 10 carbon atoms; and ##STR6##

REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part of U.S. patent application, Ser. No.370,155 filed Apr. 21, 1982 now U.S. Pat. No. 4,510,135 and U.S. patentapplication, Ser. No. 452,493, filed Dec. 23, 1982.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to pharmaceutical preparations of heparin,biologically active peptides and proteins and antineoplastic drugssuitable for enteral administration.

2. Description of the Prior Art

As a result of recent progress in the field of biochemistry, manybiologically active compounds such as heparin, proteins andantineoplastic drugs are now available for clinical use. However,because these compounds possess low lipophilicity and can be destroyedin the gastrointestinal tract by enzymes of cleavage and in the stomachby acid hydrolysis, methods of administering these compounds orally havenot kept pace with their synthesis and identification. Typical of thissituation is the case of insulin. It has long been established thatinsulin is an effective endogenous hormone useful in the treatment ofdiabetes mellitus. Furthermore, the intact insulin molecule is known topass through the intestinal wall of various animals under specifiedconditions. However, adult animals (including humans) absorb insulinpoorly when it is orally administered. This is probably due to acombination of factors: destruction of intact insulin molecules aspreviously discussed and slow passage of intact insulin moleculesthrough the intestinal wall because of low lipophilicity. Consequently,therapeutic use of insulin is limited by the necessity of administeringit parenterally, particularly by intravenous or intramuscular injection.

The desire to avoid parenteral administration of insulin has stimulatedresearch efforts in other modes of administration, among which oraladministration is the most attractive. Although efforts have been madeto develop oral hypoglycemic agents other than insulin, a great deal ofeffort has also been concentrated on the modification of insulin in sucha way that an immunologically intact and metabolically competent insulinmolecule can be absorbed through the intestine so that insulin itself ora derivative thereof may be orally administered. The search in this areahas been concentrated in three directions: the development of adjuvants,the co-administration of enzymatic inhibitors, and the development ofliposomes. Adjuvants used with insulin include resorcinols, non-ionicsurfactants such as polyoxyethylene oleyl ether, and n-hexadecylpolyethylene ether. Enzyme inhibitors include pancreatic trypsininhibitor, diisopropylfluorophosphate (DFP), and trasylol. Liposomesinclude water-in-oil-in-water insulin emulsions as well as conventionalliposomes.

The co-administration of enzyme inhibitors has had some degree ofsuccess, particularly when used with duodenal administration. Adjuvantssuch as hexylresorcinol have been administered with insulin to diabeticpatients to give systemic, hypoglycemic effects. However, some adjuvantsare limited to successful intra-jejunal administration. Compared to theother types of oral insulin preparations, liposomes have been relativesuccessful. Several studies have shown systemic, hypoglycemic effectsafter administration of a liposome containing insulin (e.g., Patel etal, FEBS Letters, 62, 60 (1976); Hashimoto et al, Endocrinol., Japan,26, 337 (1979)). However, liposomes are still in the development stageof their use as oral hypoglycemic agents and face continued problems ofstability, shelf-life, and so forth.

The difficulties of preparing other peptide and protein hormones (andother biologically active peptides and proteins) for oral ingestion orother types of enteral administration parallel the problems associatedwith insulin. Accordingly, there remains a need for a compositiongenerally capable of effecting the oral administration of biologicallyactive peptides and proteins.

Similarly it has long been established that heparin is an effective andsafe blood anticoagulant. However, therapeutic use of heparin is limitedby the need to administer it parenterally. A great deal of effort hasbeen spent on the development of adjuvants, derivatives, analogs andexpedients to render heparin absorbable from the intestine, so that itmay be orally administered. This effort includes adjuvants such asheparin coadministered with ethylenediaminetetraacetate, EDTA (Windsoret al, "Gastrointestinal Absorption of Heparin and SyntheticHeparinoids", Nature, 190, 263-264 (1961); Tidball et al, "Enhancementof Jejunal Absorption of Heparinoid by SodiumEthylenediaminetetraacetate in the Dog", Proc. Soc. Exp. Biol. Med.,111, 713-715 (1962); Rebar et al, "Forderung der GastrointestinalenResorption von Heparin durch Calciumbindungsmittel", Experientia, 19,141-142 (1963)), with dimethylsulfoxide, DMSO, and diethylsulfone, andtheir homologs (Koh, T. Y., "Intestinal Absorption of Heparin", Can. J.Biochem., 47, 951-954 (1969)); derivatives such as heparin thatunderwent partial desulfation and methylation (Salafsky et al,"Intestinal Absorption of a Modified Heparin", Proc. Soc. Exp. Biol.Med., 104, 62-65 (1960)) or heparinic acid and/or heparinic acidcomplexes (Koh et al, "Intestinal Absorption of Stable Heparin AcidComplexes," J. Lab. Clin. Med., 80(1), 47-55 (1972)); analogs (Jarrettet al, "Effect of Intravenous and Oral Administration of Heparinoids G31150, G-31150-A, and of Nitrolotriacetic Acid on Blood Coagulation",Throm. Diath Haemorrh, 25, 187-200(1971)); and expedients, such asinstillation of heparin in acidic solutions in the animal intestinalloop (Loomis, T. A., "Absorption of Heparin from the Intestine", Proc.Soc. Exp. Biol. Med., 101, 447-449 (1959); Sue, T. K., "Heparin,Physical and Biological Factors in Absorption" in "Heparin: Structure,Cellular Functions and Clinical Applications", Ed., N. M. McDuffie,Academic Press, New York, 1979, pp. 159-166). Windsor, U.S. Pat. No.3,088,868, discloses orally administrable heparin comprising heparincomplexed with the alkali metal salts of amino acids orpolyaminepolyacids, e.g., salts of EDTA. Koh et al, U.S. Pat. Nos.3,506,642 and 3,577,534, disclose heparin complexed with weakly basiccompounds (pk_(B) =7.0-12.5) being useful as an orally activemedicament. However, too highly basic materials, e.g., aliphatic amines,are taught to produce materials which are not orally active. Engel etal, U.S. Pat. No. 3,574,832, disclose a heparin composition for oral,intraduodenal or rectal administration comprising heparin and asulfate-type surfactant. Sache et al, U.S. Pat. No. 4,239,754, disclosesorally active heparin compositions comprising heparin retained on or inliposomes, the lipids of said liposomes are preferably phospholipidscomprising acyl chains derived from nonsaturated fatty acids.

While limited success has been achieved in the direction of increasingheparin absorbability from the intestine, these efforts have not yetreached the stage that heparin can be administered orally to give asustaining systemic anticoagulant effect. In short, these efforts todevelop an orally administered heparin for use in clinical anticoagulanttherapy have so far been unsuccessful.

In related work, complexes of heparin with quaternary ammonium ions suchas tridodecylmethyl ammonium chloride, TDMAC (Leininger et al, Science,152, 1625(1966); Grode et al, J. Biomed. Mater. Res. Symp., 3,77(1972)), benzalkonium chloride, BKC (Grode et al, J. Biomed. Mater. Res.Symp., 3,77 (1972); Gott, U.L., Adv. Exp. Med. Bio., 52 35 (1975)), andcetylpyridinium chloride, CPC (Schmer et al, Trans. Am. Soc. Artif.Intern. Organs, 22,654 (1976)), have been proven to render heparinsoluble in organic solvents. The heparin-surfactant complexes have beensuccessful in the coating of internal surfaces of plastic medicalappliances. Chang, U.S. Pat. No. 3,522,346, discloses the preparation ofnon-thrombogenic microcapsules wherein the encapsulating membraneincorporates or has on its surface a quaternary ammonium-heparincomplex. Suitable quaternary ammonium compounds are benzalkonium,cetyltrimethylammonium and cetyldimethylbenzyl-ammonium. Harumiya et al,U.S. Pat. No. 3,844,989, discloses antithrombogenic polymercompositions, useful in the production of medical appliances, comprisinga polymer containing cationic monomer units and heparin internally boundthereto. Grotta, U.S. Pat. No. 3,846,353, discloses a method of making anon-thrombogenic plastic material by exposing the plastic to awater-insoluble, organic solvent-soluble long chain alkyl quaternaryammonium salt having 2-4 alkyl groups and then exposing the plastic toheparin. Subsequent exposure of the plastic to blood plasma failed torelease heparin in an anticoagulant effective amount. Ericksson et al,U.S. Pat. No. 4,265,927, disloses a method of heparinizing the surfaceof a medical article by contacting the article with a complex of heparinand a cationic surfactant, preferably of the primary amine type.Marchisio et al, U.S. Pat. No. 3,865,723, discloses the use of polymerswith a polyamidic-aminic structure to remove heparin from blood.

Surfactants like BKC and CPC are cationic surfactants and widely used asantimicrobials (The Extra Pharmacocopia, Matindale, 27th Ed., ThePharmaceutical Press, London (1977)) and are quite toxic, e.g., LD₅₀ ofCPC, i.v. (mouse) is 10 mg/kg, i.v. (rat) is 6 mg/kg (Registry of ToxicEffects of Chemical Substances, U.S. Dept. HEW, 1975 Edition). Theirtoxicity is related to those various biological effects of quaternaryammonium heads whose effects include the depolarization of muscle tissueand hemolysis of erythrocytes. Toxic symptoms include dyspnoea andcyanosis due to paralysis of the respiratory muscles, possibly leadingto asphyxia (Gastmeier et al, Z. Ges. Gerich. Med., 65, 96 (1969)) andallergic reactions, after repetitive applications of quaternary ammoniumsalt solutions to the skin, which have been reported to occur in somepatients (Morgan, J. K., Br. J. Clin. Prac. 22, 261 (1969); Lansdown etal, Br. J. Derm., 86, 361(1972)). It is also believed that thesurfactant characteristics of the quaternary ammonium ion, particularlyin the liver, causes additional alterations in a number of chemical,biological and transport phenomena (Bohr et al, "Labile QuaternaryAmmonium Salt as Soft Antimicrobials", J. Med Chem. 23, 469-474 (1980)).

A related problem in biochemistry is to be able to contact tumor cellsspread along the hymphatic pathways which metastasize in the lymph nodeswith intact antineoplastic drugs and agents. Presently, such agents havebeen subject to the same problems discussed in the above discussion. Assuch contact of antineoplastics with tumor cells in the lymph nodes hasnot been very successful.

SUMMARY OF THE INVENTION

It is an object of this invention to provide an effective method oforally administering biologically active compounds including heparin,proteins and peptides and antineoplastic drugs.

It is a further object of this invention to provide compositionscontaining heparin, biologically active proteins and peptides orantineoplastic drugs which are effective when administered orally or byother enteral methods.

It is still a further object to provide a method of producing suchcompositions which can be carried out with heparin, biologically activepeptides or proteins and antineoplastic drugs.

It is yet a further object to provide a covalent compound which permitscontact with tumor cells located in the lymph nodes.

In accordance with the present invention a complex of heparin with aquaternary ammonium ion selected from the group consisting of ##STR7##where

R¹ and R² are the same or different and are alkyl or hydroxy substitutedalkyl; and

R" is a saturated or unsaturated aliphatic containing at least 10 carbonatoms; ##STR8##

where R¹, R² and R" have the meanings given above; and R and R' aresaturated or unsaturated aliphatics containing at least 10 carbon atoms;##STR9##

where R¹ and R² have the meanings given above; R³ is independently alkylor hydroxy substituted alkyl containing at least 10 carbon atoms; and##STR10##

where R¹, R², R³ R and R' have the meanings given above.

In further accordance with the present invention a composition isdisclosed comprising

a sandwich complex comprising a hydrophobic core complex of abiologically active peptide or protein with an alkyl or alkenyl sulfatehaving 6-24 carbon atoms and 0-3 double bonds which forms anelectrostatic complex with a quaternary ammonium ion selected from thegroup consisting of ##STR11## where

R¹ and R² are the same or different and are alkyl or hydroxy substitutedalkyl; and

R" is a saturated or unsaturated aliphatic containing at least 10 carbonatoms; ##STR12##

where R¹, R² and R" have the meanings given above; and R and R' aresaturated or unsaturated aliphatics containing at least 10 carbon atoms;##STR13##

where R¹, R² and R³ have the meanings given above; and ##STR14##

where R¹, R², R³ R and R' have the meanings given above.

In further accordance with the present invention a compound is set forthselected from the group consisting of ##STR15##

where therapeutic agent is heparin, a biologically active peptide orprotein or an antineoplastic drug; and ##STR16## where

therapeutic agent has the meanings given above; and

R and R' are the same or different and are saturated or unsaturatedaliphatic containing at least 10 carbon atoms.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention involves a method of modifying a biologicallyactive compound in such a way that the compounds are absorbed into thesystemic circulation when administered enterally, particularly orally,while remaining immunologically intact and metabolically competent. Toachieve this result, the biologically active compound is coupled to aprotective carrier in the form of a complex or covalent compound. To besuccessful the complex or compound must meet the following criteria: (a)it must be resistant to the acidic environment in the stomach; (b) itmust be resistant to enzymatic degradation by gastric and pancreaticenzymes; (c) it must be sufficiently lipophilic to pass the intrinsicbarrier of the intestinal wall; and finally (d) the changes inphysiological and biological properties of the biologically activecompound resulting from the modification must be minimal so that theirhormonal activity is maintained. While criteria (c) and (d) are basicstructural requirements for all enterally administered compounds (suchas by rectal, buccal or topical routes), criteria (a) and (b) must bemet in addition to (c) and (d) for the compound to be orally effective.

Specifically, in one preferred embodiment, it has been discovered thatheparin forms complexes with certain "soft" or "pseudo" quaternaryammonium ions to render heparin hydrophobic and lipophilic to therebycarry heparin through the lipid barrier of the intestinal wall and,consequently, increase the absorbability of heparin from the intestine.A systemic anticoagulant effect can thereby be achieved.

It will be understood that heparin is a very complex molecule, with astructure which has not been completely elucidated. It is tentativelyidentified as a sulfated copolymer consisting of alternating 1-4 αlinked glucosamine and glucuronic acid residues. In accordance with theinvention, heparin is combined with certain quaternary ammonium ions,which is themselves are not simple. Therefore the specific structure ofthe resulting product cannot be stated with certainty and theterminology "complex" is used to embrace the structures which may beformed. Preferably, the complex contains five moles of the ammonium ionof this invention per mole of heparin tetrasaccharide unit, it beingbelieved that complexation occurs with the sulfate groups of thetetrasaccharide unit. For purposes of illustration a schematicrepresentation of the repeating heparin tetrasaccharide unit (sodiumsalt) is shown below ##STR17##

Specifically, complexes of heparin are formed with quaternary ammoniumions selected from the group consisting of ##STR18## where

R¹ and R² are the same or different and are alkyl or hydroxy substitutedalkyl containing 1 to 6 carbon atoms; and

R, R' and R" are the same or different and are saturated or unsaturatedaliphatics containing at least 10 carbon atoms ##STR19##

where R¹, R², R and R' have the meaning of ion (I) and R³ isindependently an alkyl or hydroxy substituted alkyl containing 1 to 6carbon atoms: ##STR20##

where R¹, R² and R" have the meanings given above and ##STR21##

where R¹, R² and R³ have the meanings given above.

The present invention also provides a sandwich-type complex of abiologically active peptide or protein with an alkyl sulfate and a softquaternary ammonium ion. The biologically active peptide (hereafter"peptide" or "protein" will refer to both peptide and protein moleculesunless otherwise indicated) first forms a hydrophobic core complex withthe alkyl sulfate. This core complex protects the peptide molecule fromacidic hydrolysis and enzymatic degradation and increases thelipophilicity of the peptide molecule, thereby allowing the intactpeptide to pass through the stomach when orally ingested and to increasethe rate at which it is absorbed through the intestine wall. This innercomplex may be formed between the peptide molecule and the alkylsulfate. Hydrophobic complexes of a peptide with an alkyl sulfate aregenerally rod-like with a helical polypeptide chain of the peptideexisting within a hydrophobic shell formed by the alkyl sulfate. Typicalof alkyl sulfate complexes with protein are those complexes formed withsodium dodecyl sulfate (SDS). SDS has been found to bind proteinmolecules in constant gram to gram ratios irrespective of nature ofprotein but depending on the SDS monomer concentration. When SDS monomerconcentration exceeds 5×10⁻⁴ M, proteins form complexes with SDS in ahigh binding ratio where one gram of protein binds to about 1.4 gram ofSDS. When the SDS momomer is less than 5×10⁻⁴ M, proteins form complexeswith SDS in a low binding ratio where one gram of protein binds to about0.4 gram of SDS. Both protein SDS complexes assume a similar, rod-likeshape with a helical polypeptide chain or protein folded back uponitself near its middle to give a double helical rod and the SDS forminga shell about the rod via hydrophobic forces. The sulfate groups of SDSare on te surface of the rodlike complexes as evidenced by theelectrophoretic migration of insulin in the presence or in the absenceof SDS. In electrophoresis at pH3 in the absence of SDS, insulin isfully protonated and migrates to cathode. In the presence of SDS (0.1%)at pH 3, insulin migrates to the anode (as if it is an anion).

Since alkyl sulfates are themselves hydrolyzed to fatty acid alcoholsand a sulfuric acid salt at acidities approximately those of thestomach, the rod-like peptide alkyl sulfate complex requires anadditional protective coating for oral administration which is providedby a soft quaternary ammonium ion, the structures of which are describedlater in detail.

By utilizing the method of the invention, it is possible to preparepeptide compositions suitable for oral administration which containendogenous opioid agonists, such as encephalins and endorphins;hypothalmic hormones, such as gonadoliberin, melanostatin,melanoliberin, somatostatin, thyroliberin, substance P, and neurotensin;adenohypophyseal hormones, such as corticotropin, lipotropin,melanotropin, lutropin, thyrotropin, prolactin, and somatotropin;neurohypophyseal hormones; calcitropic (thyroid) hormones, such asparathyrin and calcitonin; thymic factors, such as thymosin,thymopoietin, circulating thymic factor, and thymic humoral factor;pancreatic hormones, such insulin, glucagon, and somatostatin;gastrointestinal hormones, such as gastrin, cholecystokinin, secretin,gastric inhibitory polypeptide, vasointestinal peptide, and motillin;chorionic (placental) hormones, such as choriogonadotropin andchoriomammotropin; ovarian hormones, such as relaxin; vasoactive tissuehormones, such angiotensin and brandykinin; growth factors, such assomatomedins, epidermal growth factor, urogastrone, and nerve growthfactor; hemophilia factors, such as blood clotting factors VIII and IX;enzymes, such as streptokinase, fibrinolysin, deoxyribonuclease, andasparaginase; and artificial or pseudo peptides, such as deferoxamine.Many other classes and specific types of peptide and protein hormonesand other biologically active molecules are known. Peptide and proteinhormones suitable for use in the present invention are disclosed inJohannes Meienhofer, "Peptide and Protein Hormones", in Burger'sMedicinal Chemistry, 4th ed., (part II), Wolff, Ed., John Wiley and Sons(1979), which is herein incorporated by reference. Preferred hormonesare those with a molecular weight of less than 7000, with insulin beingespecially preferred.

The listings of peptides and proteins in this application are notintended to be exclusive, and it may easily be determined by simpleexperimentation if any protein having biological activity can beprepared into a complex according to the invention. One simple method oftesting for core complex formation involves the following steps: (1)dissolve approximately 10 mg of the biologically active peptide orprotein in a small amount of water or buffer; (2) adds about 15 mg of analkyl sulfate, for example sodium dedecyl sulfate, mix well and allow tostand for about 5 minutes; (3) subject the resulting solution to agaroseor acrylamide gel electrophoresis. The complex acts as an anion even atlow pH (about 3 is a good testing point) because of the sulfate groupsand migrates toward the anode. If no complex has formed, the proteinwill be protonated at low pH and migrate toward the cathode.

If complex formation has taken place and if the resulting core complexwill itself complex with a soft quaternary ammonium ion according to theprocess of the present invention (infra), the biologically activepeptide is suitable for use in the present invention.

Complex formation between protein and a carrier molecule is one way toprotect a protein molecule from acidic hydrolysis and enzymaticdegradation and to increase the lipophilicity of the protein molecule.Depending on the carrier substances selected, formation of this complexcan be via columbic interaction between protein molecule and carriersubstance or via hydrophobic forces. In the proposed sandwich complex ofsulfates, e.g., sodium dodecyl sulfate, are commercially available.Suitable examples of preferred linear alkyl sulfates include octylsulfate, nonyl sulfate, decyl sulfate, undecyl sulfate, dodecyl sulfate,tridecyl sulfate, tetradecyl sulfate, pentadecyl sulfate, and hexylsulfate. Of these, decyl sulfate, dodecyl sulfate and tetradecyl sulfateare more preferred with dodecyl sulfate being most preferred.

The alkyl sulfates are generally present initially as alkali metal saltswhen the initial core complex is being formed. Alkali metal saltsinclude lithium, sodium, potassium, rubidium and cesium salts. Of these,sodium and potassium are preferred, with sodium salts being mostpreferred. Sodium and potassium salts of dodecyl sulfate are especiallypreferred, with sodium dodecyl sulfate being the most preferred alkylsulfate salt.

The weight ratio of protein or peptide to alkyl sulfate is the weightratio of the naturally forming complex. In a preferred embodiment,insulin is complexed with sodium dodecyl sulfate (SDS). This complexforms as insulin:SDS complex in a ratio of 1:1.4 or 1:0.4 by weight(depending on the initial ratio present) and is a hydrophobic complex.Complexes of protein with SDS are preferred to other types of complexesbecause a wide variety of proteins are reported to bind to an identicalsulfates, e.g., sodium dodecyl sulfate, are commercially available.Suitable examples of preferred linear alkyl sulfates include octylsulfate, nonyl sulfate, decyl sulfate, undecyl sulfate, dodecyl sulfate,tridecyl sulfate, tetradecyl sulfate, pentadecyl sulfate, and hexylsulfate. Of these, decyl sulfate, dodecyl sulfate and tetradecyl sulfateare more preferred with dodecyl sulfate being most preferred.

The alkyl sulfates are generally present initially as alkali metal saltswhen the initial core complex is being formed. Alkali metal saltsinclude lithium, sodium, potassium, rubidium and cesium salts. Of these,sodium and potassium are preferred, with sodium salts being mostpreferred. Sodium and potassium salts of dodecyl sulfate are especiallypreferred, with sodium dodecyl sulfate being the most preferred alkylsulfate salt.

The weight ratio of protein or peptide to alkyl sulfate is the weightratio of the naturally forming complex. In a preferred embodiment,insulin is complexed with sodium dodecyl sulfate (SDS). This complexforms an insulin:SDS complex in a ratio of 1:1.4 or 1:0.4 by amount ofSDS on a gram per gram basis. See, for example, Reynolds et al., Proc.Nat. Acad, Sci. (US), 66, 1002-1003 (1970) and Reynolds et al., J. Biol.Chem., 245, 5161-5165 (1970). When SDS forms a complex with insulin orother protein, the hydrophobic core complex is rod-like with a helicalpolypeptide chain of protein existing within a hydrophobic shell formedby the SDS. This complex of protein with alkyl sulfate is referred toherein as a core complex. This term is not intended to limit the presentinvention, but is believed to be generally descriptive. When this corecomplex is itself complexed with a soft quaternary ammonium ion, anadditional layer forms on the surface of the inner complex. This lattercomplex is referred to as an "electrostatic" complex. Nevertheless, thisterm additionally is not intended to be limiting of the actual physicalstructure that is present in the resulting complex.

As mentioned above, the inner complex is reacted with quaternaryammonium ion to form an outer complex. The phrase quaternary ammoniumion as used in this invention includes the ions having the structuralformulas I-IV where R¹, R², R³, R, R' and R" have the meanings givenearlier.

The present invention is also directed to a new compound whichincorporates a therapeutic agent, which may be heparin, a biologicallyactive peptide or protein or antineoplastic drug bound to a triglyceridetype backbone structure by covalent bond to form a compound which isenterically introduced into the body. This compound, like the complexesdiscussed above, provides a means of providing an intact bioactivematerial, of the types mentioned earlier into the body.

A particular advantageous feature of this embodiment is the ability totarget antineoplastic drugs and agents into the lymphatic system. Asthose skilled in the art are aware, tumor cells often spread along thelymphatic pathways and metastasize in the lymph nodes. Thus, in apreferred embodiment the therapeutic agent covalently bonded to thetriglyceride structure is an antineoplastic agent. For example,1-asparaginase can be bonded to the triglyceride type backbone.

A triglyceride structure is covalently bonded to the therapeutic agentbecause it is known that the digestion of triglycerides (fat) in thelumen of intestine proceeds first by the removal of a terminal fattyacid to produce a 1,2-diglyceride. The other terminal fatty is thenremoved to produce a 2-monoglyceride. As pancreatic lipase is virtuallyspecific for the hydrolysis of primary ester linkage, to remove thefatty acid from 2-monoglyceride requires its isomerization to primaryester linkages, i.e., 1-monoglyceride. The isomerization is a relativelyslow process, as a result 2-monoglyceride is the major end product offat digestion and less than 25% of the ingested fat is completely brokendown to glycerol and fatty acid. Within the intestinal mucosa,2-monoglyceride and some of 1-monoglyceride may be reconverted totriglyceride, whereas about 22% of 1-glycerides are hydrolyzed toproduce free glycerol and free fatty acid which are passed directly tothe portal vein. The triglycerides so synthesized will be incorporatedin the form of chylomicrons which are transported into the lymphatics(the lacteals) and hence via thoracic duct into the circulating blood.

The triglyceride type backbone to which the therapeutic agent iscovalently bonded results in a structural formula selected from thegroup ##STR22##

where R and R' are the same or different and saturated or unsaturatedaliphatics containing at least 10 carbon atoms; and therapeutic agentmay be heparin, a biologically active peptide or protein or anantineoplastic agent; and ##STR23##

where therapeutic agent has the meanings given above.

Another aspect of the present invention is the ammonium ions complexedwith heparin or a biological peptide or protein. One of these ammoniumions is the ion having the structural formula III. This ion isformulated by reacting glyceride with benzaldehyde is toluene in thepresence of p-toluene sulfonic acid catalyst. This reaction, preferablyby refluxing the reactants for about 5 hours, produces a mixture of1,2-benzylidene glycerol, an oil and 1,3-benzylidene glycerol, a solid.The 1,3-benzylidene glycerol crystals are separated from the1,2-benzylidene glycerol liquid.

In an alternative method, 1,3-benzylidene is formed in the absence ofany other product, simplifying its recovery. In this process stephydroxyacetone is reacted with benzaldehyde using toluene solvent in thepresence of a catalytic amount of p-toluene sulfonic acid.

As in the first reaction, the reaction is conducted at reflux over aperiod of about 5 hours. The product of this reaction is5-oxo-2-phenyl-1,3-dioxane. In turn, the dioxane is reacted with sodiumborohydride and then the reaction product is reacted in-situ with sodiumhydroxide which results in the formation of 1,3-benzylidene glycerol.

Independent of the method utilized to synthesize the 1,3-benzylideneglycerol it is reacted with potassium metal to produce the potassiumsalt of 1,3-benzylidene glycerol oxide. The reaction is conducted in axylene slurry of the finely divided potassium metal under reflux andstirring. The oxide is reacted with an excess of dimethylaminoethylchloride under reflux and stirring for at least 2 hours. Upon coolingthe byproduct, potassium chloride, is removed by filtration. Uponpurification 1,3-benzylidene glycerol ether is formed.

The 1,3-benzylidene glycerol ether in 1,3-N,N-dimethylbenzylidene isreacted with an alpha-chloroethyl ester of a long chain saturated orunsaturated fatty acid, RC(O)OCH(CH₃)Cl, in petroleum ether or othersuitable solvent. The reaction is conducted under a blanket of drynitrogen gas with the reactants present in equimolar amount. The productof this reaction is 1,3-benzylidene glycerol ester. The reaction occursunder reflux and stirring.

The thus formed ester is reacted with 2-methyoxyethanol in the presenceof boric acid (finely divided solid) for between 1/2 and 1 hour. Thepurified product of this reaction is compound III.

To obtain the ion having the structural formula I, dihydroxy acetone isreacted with acid chloride of the desired fatty acid R--C(O)Cl in achloride acceptor, preferably pyridine, and in chloroform solution. Thisreaction results in the product, diacyl acetone having the structuralformula ##STR24## Reaction with sodium borohydride, followed by thein-situ reaction with sodium hydroxide produce 1,3-diacycl glycerol.

1,3-Diacyl glycerol, ##STR25## is then treated in accordance with theprocedures enumerated above to produce the ion having the structuralformula I.

The synthesis of the ammonium ion having the structural formula IV whereR¹, R² and R³ are each methyl is obtained by reacting 1,3-benzylideneglycerol, obtained by the procedures enumerated above, with phosphorusoxychloride in the presence of quinoline solution. The solution is waterand alcohol free with chloroform having the preferred solvent. Thereaction is conducted at slight elevated temperature, preferably about35° C., for about 1 hour to produce phosphatilic acid chloride.Preferably, the reactants are provided in equimolar amounts.

The phosphatidic acid chloride is esterified by reaction with cholineiodide, the iodide present in slight stoichiometric excess, in thepresence of pyridine to yield 1,3-benzylidene glycerophosphoryl choline.The benyzlidene protecting group is removed by heating the1,3-benzylidene glycerophoryl choline with powdered boric acid in2-methoxyethanol to yield the desired 2-glycerophosphoryl cholineiodide. Analogous procedures are used to produce the desired ammoniumion where R¹, R² and R³ are alkyl or hydroxyalkyl other than methyl.Also, halides other than iodide may also be utilized.

The same procedure is utilized to form ammonium ion having thestructural formula II except that the 1,3-diacyl glycerol, obtainable bymethods set forth earlier, is substituted for 1,3-benzylidene glycerol.

Another aspect of the present invention involves the synthesis of thecompound having the structural formula V. In this procedure 1,3-diacylglycerol is reacted with cyanogen bromide in a polar solvent, preferablydimethyl formamide, the bromide in aqueous solution in slightly basicmedium, pH of about 8.5 to produce 1,3-diacyl-2-cyano ester which reactswith the amine of the therapeutic agent to form the compound whosestructural formula is V. Those skilled in the art will appreciate thatthe compound formed has the structural formula ##STR26## However, forsimplicity the structure of formula V has been adopted.

Obviously, the analogous compound having the structural formula VI isformed by the replacement of the starting material 1,3-diacyl glycerolwith glycerol. It should also be appreciated that the actual structuralformula of V! is analogous to formula VII. For simplicity, the structureVI has been adopted.

The heparin complexes of the present invention are formed by bringingheparin into contact with the desired quaternary ammonium ion. Forexample, an aqueous solution of heparin sodium salt is mixed with thequaternary ammonium salt, preferably the halide to giveheparin-quaternary ammonium ion complexes and sodium halide.Consequently, heparin in the heparin complex is a mucopolysaccharidechain with negative sulfate groups in it associated with the positivequaternary ammonium groups, but still with sodium associated with thecarboxylic acid groups. As noted above, the reaction may be convenientlyperformed in aqueous media. The reaction conditions are quite simple,i.e. mixing aqueous solutions of the appropriate quantity of heparinsodium salt and the quaternary ammonium salt at room temperature,preferably five moles of quaternary ammonium salt are used per mole ofheparin tetrasaccharide unit. The product may be readily recovered bylyophilization, since sodium halide, the other product resulting fromthe above reaction, possesses no adverse effect when it isco-administered orally with the heparin complex. Alternately, theheparin-quaternary ammonium ion complex may be separated from sodiumhalide by gel filtration chromatography with water as the elutionsolvent. Lyophilization of the elution pool that contains the complexgives the desired purified product.

The heparin complexes of the present invention can be orallyadministered, per se, for example, as a lyophilized powder, or incombination with a pharmaceutically acceptable excipient, e.g., withwater as a dispersion or in the form of a tablet, and exhibit asustained systemic anticoagulant effect. Exemplary excipients includeinert fillers and binders such as lactose, sucrose, dextrose, sorbitoland cellulose products; disintegrating agents such as starch and soypolysaccharide; and lubricants such as magnesium stearate, stearic acidand hydrogenated vegetable oils.

Additionally, encapsulation of the complex in an enteric coating, bymeans well-known in the art, provides a suitable form for oraladministration.

The heparin complexes of the present invention may also be administeredin the form of a vaginal or rectal suppository, cream or ointment. Theformulation of such suppositories, creams and ointments can beaccomplished by techniques well known in the art.

Typically, the heparin complexes of this invention are used in amount toprovide a dosage of 1400 units of heparin per kg of body weight every 8to 12 hours. This dosage being sufficient to exhibit a sustainedsystemic anticoagulant activity.

It has also been found that when the heparin complexes of the presentinvention are prepared with a stoichiometric excess of quaternaryammonium ion being present in the final product, the ease of absorptionof heparin through the intestine is increased. Typically, the heparincomplex will contain between five moles of quaternary ammonium ion permole of heparin tetrasaccharide unit and three parts by weight ofquaternary ammonium ion per one part by weight of heparin.

Unlike the surfactants of the prior art, the above-noted formulationsare "soft" or "pseudo" quaternary ammonium ions. That is, they will bedeproponated in vivo as in the formula I ions, and/or hydrolyzed in vivoto give protonated triethyl amine, a fatty acid and an aldehyde moleculeas in the formula II ions. Consequently, they are non-toxic andeminently suited for oral administration.

Turning to the biologically active peptide and protein complexes of thepresent invention these are formed by reacting an ammonium ion havingone of the structural formulas I-IV with the alkyl sulfate in an amineto alkyl sulfate ratio of 1:0.3 to 1:1 with a 1:1 molar ratio preferred.

Since a soft quaternary ammonium ion is a postively charged reagent andsince the alkyl sulfate is a negatively charged reagent, a 1:1 molarratio of the soft quaternary ammonium ion with the alkyl sulfate ispreferred. However, other ratios are possible and fall within the scopeof the invention. Ratios of amine to alkyl sulfate in the range from1:0.3 to 1:1 are contemplated by the present invention. Such ratios areobtained by using an excess of the core complex or the amine/ammoniumion component during formation of the electrostatic complex.

The final electrostatic peptide or protein complex is formed by addingthe soft quaternary ammonium ion to an aqueous solution containing aprotein.alkyl sulfate complex (i.e., the core complex). The resultingelectrostatic complex comprising the entire protein.alkylsulfate:quaternary ammonium ion complex can be isolated by extractingthe aqueous solution with chloroform or another non-polar solventimmiscible with water. The presence of protein in the extracted complexcan be verified using the Fluorescamine protein test as described inUdenfried et al, Science, 178, 871-872 (1972), which is hereinincorporated by reference. When working with a previously untriedcomplex, this allows easy verification of the formation of the desiredcomplex.

This invention may be carried out either by preparing a pharmaceuticalcomposition which may be stored in that form or by producing thesandwich complex immediately prior to administration. When a protonatedamine is used to form the final complex, the complex can be stored atapproximately 4° C. in 0.005M phosphoric acid for at least 2 weeks. Whena soft quaternary ammonium ion is used, the complex should be preparedin deionized water at a pH of approximately 7. It can then belyophilized and stored in powder form for at least several months. Sinceoral administration is the principal contemplated end use of thecompositions of the present invention, compositions suitable for oralingestion are preferred storage forms. Such compositions can containpharmaceutically acceptable carriers in addition to the previouslydisclosed ingredients. Suitable pharmaceutical carriers include liquidor solid carriers of pharmaceutically acceptable or otherwise inertmaterials which may be used orally. Examples of liquids are water andaqueous solutions of non-toxic salts, such as sterile physiologicalsolutions of saline, or aqueous solutions containing non-toxic organicsolvents, such as ethanol, used to increase the amount of complex insolution. Dilute aqueous solutions of mineral acids having a pH of lessthan 4 are also suitable. Phosphoric, sulfuric, and hydrochloric acidsare preferred. Also suitable are emulsions, such as oil-in-wateremulsions. Solutions of non-toxic organic liquids, such as ethanol, arealso suitable. Solid carriers include both nutritive carriers, such assucrose or gelatin, and non-nutritive carriers, such as cellulose ortalc. A pharmaceutical preparation of the invention may be in the form,for example, of a liquid, a capsule, a tablet, or a suppository.

Pharmaceutical compositions according to the present invention areadministered in dosages which depend upon the effect desired for thebiologically active compound which is being administered. Thedetermination of the effective amount of the biological compound is notconsidered to be part of the present invention since dosage rates aregenerally determined by the effect of the composition on the particularpatient taking the medication. An amount equal in dose rate (mg/kg) tothe amount normally injected parenterally for known biologically activepeptides is suitable for use in the present invention as an initial doseand may be adjusted as necessary to achieve the desired effect.

A particularly preferred embodiment of this invention comprisesenterally administering a sandwich complex of the invention containinginsulin as the active ingredient to produce a hypoglycemic effect. Theamount required will depend on the severity of the diabetes and on thecondition of the patient (e.g., time since ingestion of food, type andamount of food ingested, etc.). Adjustment of the amount required tomaintain the proper blood glucose level is within the capability ofthose of ordinary skill in the art. An orally administered sandwichcomplex (of insulin or any other active peptide) is especiallypreferred.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific examples which are provided herein for purposes of illustrationonly and are not intended to be limiting unless otherwise specified.

EXAMPLE 1 Synthesis of 1,3-Benzylidene glycerol

A mixture of glycerol (15 g, 0.163 mole) and a slight excess ofbenzaldehyde (18.7 g, 0.175 mole) in 60 ml of toluene with a catalyticamount of p-toluenesulfonic acid (0.3 g) was refluxed and stirred for 5hr. in a Soxhlet apparatus containing dry MgSO₄ for removal of water.The products, a mixture of 1,3 and 1,2-benzylidineglycerols was obtainedby distillation under reduced pressure. The reaction products wereseparated on a Florisil column (1.5×42 cm) and eluted withn-hexane:ether 90:10 (100 ml), 70:30 (350 ml), 50:50 (450 ml) and 0:100(300 ml). The desired product 1,3-benzylideneglycerol was derived as acrystalline product in 70:30, 0:100 elution, the only product of the50:50 eluting solvent was the 1,2 isomer. The 1,3-benzylideneglycerol(MP 50°-60° C.) was further purified by recrystallizing the combinedcrystalline products from benzene-petroleum ether (m.p. 82°-83° C.).

EXAMPLE 2 Synthesis of 1-3-benzylidene glycerol

The procedure of Example 1 was repeated except that dihydroxyacetonereplaced glycerol. The resultant product of this reaction was5-oxo-2-phenyl-1,3-dioxane. Since the dioxane is the sole product ofthis reaction no column separation was required. The dioxane was reducedto 1,3-benzylidene glycerol by reaction with sodium borohydride followedby in-situ reaction with sodium hydroxide.

EXAMPLE 3 Synthesis of 1,3-benzylidene glycerol ether

1,3 Benzylideneglycerol formed in Example 1 or 2 was added in portionsat 1:1 mole ratio to vigorously stirred, finely powdered potassium inxylene (0.1 mole of reaction mixture in 300 ml of xylene) (52) and thereaction continued with refluxing and stirring. Excessdimethylaminoethyl chloride (0.175 mole) was added in portionsaccompanied by refluxing and stirring. This reaction continued for 20hours. After cooling, potassium chloride was removed via filtration andthe filtrate was evaporated under reduced pressure to remove xylene andunreacted dimethylaminoethyl chloride. The residue was distilled toremove the unreacted 1,3-benzylideneglycerol. The1,3-benzylideneglycerol ether was obtained by crystallizing the residuefrom ethanol.

EXAMPLE 4 Synthesis of the Ammonium ion having Structural Formula I

An appropriate amount α-chlormethyl ester [RC(O)OCH₂ Cl] (or-chloroethyl ester [RC(O)OCH(CH₃)Cl]) of a saturated (or unsaturated)fatty acid having 18 carbon atoms using the method of Bohr et al (J.Med. Chem 23, 469-474, 1980) was added dropwise in a 1:1 mole ratio to avigorous stirred solution of1,3-N,N-dimethylbenzylideneglycerolethylamine in dry petroleum etherunder a nitrogen atmosphere. Refluxing and stirring was continued untilthe reaction was complete. The solvent was then evaporated under reducedpressure, and the desired products were obtained either via columnchromatography (florisil) or crystallization from the appropriatesolvent.

The benzylidene blocking group of the desired product was removed byheating the benzylideneglycerol ester (0.01 mole) and finely powderedboric acid (0.1 mole) at a 1:10 mole ratio in an appropriate volume of2-methoxy ethanol, ca. 20 ml, for 30 min to one hr. A small amount ofboric acid remained undissolved. The mixture was dissolved in ether andwashed with water, then the ethereal solution was dried (Na₂ SO₄). Theresidue was isolated and purified via crystallization from ether.

EXAMPLE 5 Synthesis of 2-glycerol phosphoryl chloride

1,3-benzylideneglycerol, obtained in Example 1 or 2 was phosphoratedwith phosphorus oxychloride (one molar equivalent) in the presence ofquinoline. The resulting phosphatidic acid chloride was esterfied withcholine iodide (1.125 molar equivalent) in the presence of pyridine. Thereaction mixture was separated by column chromatography on silicic acidto give 1,3-benzylideneglycerophosphoryl choline. The benzylideneprotecting group was removed by heating the above product with powderedboric acid in 2-methoxyethanol to give the desired 2-glycerolphosphorylcholine iodide.

EXAMPLE 6 Thoracic Duct Cannulation of Rats

Three rats were fed 1 ml of corn oil via gavage. Three hours later,thoracic duct cannulation and feeding duodenostomy was performed underether anesthesia. The body temperature of the rats were maintained at36°-38° C. during and after the operation. The thoracic duct fistulaconsisted of three parts, i.e. the main body--a 30 cm PE 160 tubing (id1.14 mm, od 1.57 mm) with a U-shape bent at one end, (the short arm isabout 1 cm long), a 7.5-5 mm tip made of PE 60 tubing (id 0.76 mm, od,1.22 mm) and a 30 cm of PE 10 (id, 0.28 mm, od, 0.61 mm) inserted at thebend and glued onto the PE 160 tubing as a side arm for a slow,continuous infusion of anticoagulant solution.

Cannulation of the thoracic duct was accomplished through an incisionjust distal to the last rib, extending from the midling anteriorly ofthe left quadratus lumboram muscle posteriorly. A small gauze packwetted with isotonic saline was placed such that it pushed the stomach,liver, and intestine back and to the right, exposed the diaphragm, theaorta, and the left adrenal gland and kidney. Retractors were used toretract the kidney distally and to hold the incision open. With the useof a blunt, curved microforcep, a small opening was made in theperitoneum over the quadratus lumborum, approximately 0.5 cm cephalad tothe superior suprarenal artery. The vein and peritoneum were dissectedsuperficially and retracted to the right until the left subcostal arterywas exposed. The thoracic duct was visible just posterior to the aorta,appeared to be opaque due to the presence of corn oil. It was 1 to 2 mm.in diameter and was embedded in loose connective tissue and fat. Thethoracic duct was exposed for a length of 5 to 8 mm by gently bluntdissection, and was ligated at the upper end of the exposed portion just3 to 5 mm cauded to the subcostal artery. After one or two minutes, theportion of thoracic duct at the point of ligation was expanded orballooned with a milky lymphatic liquid, the sharp tip of the thoracicfistula was then inserted into the swelling and the opaque lymph fluidimmediately filled the fistula. The fistula was held in place with acyanoacrylate adhesive.

The duodenal cannula comprised a PVC tubing (20 cm long, with id 1.10 mmand of 1.50 mm) with a 3-5 mm sleeve which was made of tycon tubing (id1.56 mm, od 3.13 mm) and glued at one end of the tube. The feedingduodenostomy was accomplished by inserting the cannula at the sleevedend in a distal direction through the stomach and into the duodenum. Theduodenal cannula was tied firmly in position with purse string switch;and the tycon sleeve further helped the cannula stay within thegastroduodenal tract.

EXAMPLE 7

At the end of the operations of Example 6, the skin incisions wereclosed. Body temperature of the rats were maintained at 36°-38° C.during and after the operation.

Postoperatively, the rats were placed in restraining cages overnight andgiven free access to an aqueous solution containing dextrose 100 g, NaCl5 g, and KCl 0.4 g/l. An amino acid/carbohydrate mixture (Vivonex, 0.5strength, Eaton Laboratories) was pumped into the duodenum at 3 ml/hr.Anticoagulant solution (sodium citrate) was also pumped into thethoracic fistula via the PE 10 side-arm at 0.8-1 ml/hr. If the lymphflow was less than 2 ml/hr, the rat was discarded.

At 12 to 18 hours postoperative, a bolus dose of 2-monoglycerolphosphoryl choline.tritiated haparin complex at a dosage 1/2 to 2/3 thatorally administered in Comparative Example 1 was pumped into theduodenum. Afterwards, the pumping of Vivonex into the duodenum resumedat 3 ml/hr. Lymph fluid was continuously collected for 24 hours using aGilson fraction collector at 30 minutes per fraction. The fractions werecounted in a xylene in a xylene-based scintillant (Instagel, PackardInst.). The lymph anticoagulant activity was determined by the activatedpartial thromboplastin time (APTT) test. This test determines the timefor recalcified plasma to clot after incubation with a reagentcomprising a platelet phospholipid and a contact activator which is ameasure of the rate at which thrombin is formed via the intrinsicpathway of coagulation. For up to 24 hours anticoagulant activity wasobserved. Actual coagulant effect on the three rats operated on inaccordance with Example 6 is summarized in Table I below.

The rats were sacrificed after 24 hours. The intestine with mesentaryand lumenal content, from the esophagus to the rectum including thestomach, was removed and homogenized (GI tract homogenate). The spleen,liver, kidney, heart and lung were homogenized individually. Aliquots ofeach homogenate (digested in NC5 Amersham/Searle) were counted in ascintillant containing PPO (2,5-diphenyl oxazole) (6 g) and POPOP(1,4-bis[2-(5-phenyl oxazolyl)benzene], (75 mg)/liter of toluene.

The amount of tirtuated (³ H) heparin complex (HC) infused (D) is equalto the dosage given.

The amount of ³ H-heparin complex (HC) infused (D) is equal to thedosage given. The amount of ³ H activity in the lymph (HC_(lym)) wasmeasured directly as is that remaining in the GI Tract homogenate(HC_(gi)). It is assumed that ³ H-heparin complex, which is absorbed butnot recovered, is transported via the portal venous circulation.##EQU1##

From this data the % heparin complex transported in the lymph and %heparin complex remaining in the gastro-intestinal tract is determined.This data is included in Table 2.

                  TABLE 1                                                         ______________________________________                                        Rat No.      155         160     161                                          ______________________________________                                        Rat Wt., g   420.8       413     291                                          Dosage, mg/kg                                                                              5.76        12.5    12.5                                         Lymphatic Fluid Anticoagulant Activity (units/ml)                             Time After                                                                    Administration                                                                 0.5         0.0761      0.0682  0.0495                                        1.0         .1230       .1972   .1150                                         1.5         .0949       .2601   .1500                                         2.0         .1695       .2643   .1960                                         2.5         .1602       .3089   .1960                                         3.0         .1879       .3628                                                0-                                                                             3.5         .1136       .1972   .1090                                         4.0         .0977       .1972   .0495                                         4.5         .0949       .2685   .2810                                         5.0         .0761       .4668   .1555                                         5.5         .1695       .3015   .0793                                         6.0         .2337       .2809   .921                                          6.5         .1136       .3089   .0615                                         7.0         .1695       .3506   0                                             7.5         .1416       .3262   0                                             8.0         .0191       .3311   .0675                                         8.5         .2791       .2643   .0615                                         9.0         .2337       .2435   .0195                                         9.5         .2610       .2435   .0075                                        10.0         .2790       .3506   0                                            10.5         .2790       .4311   .1206                                        11.0         .2337       .4311   .0795                                        11.5         .2610       .2850   .1265                                        12.0         .1230       .2850   .1090                                        12.5         .2610       .3830   .0971                                        13.0         .1250       .4826   .1355                                        13.5         .2063       .3850   .1380                                        14.0         .3061       .4232   .1670                                        14.5         .2155       .3425   .0971                                        15.0         .2701       .3180   .1555                                        15.5         .2791       .3056   .0375                                        16.0         .4108       .2183   .2360                                        16.5         .4108       .3871   .0075                                        17.0         .1695       .3261   0                                            17.5         .2063       .7486   .2360                                        18.0         .1695       .2225   .0855                                        18.5         .1230       .1845   0                                            19.0         .0572       .3256   .3475                                        19.5         ND          .3830   .3200                                        20.0         ND          .6265   .2530                                        20.5         ND          .5256   .3090                                        21.0         ND          .3871   .2416                                        21.5         ND          .4311   .0971                                        22.0         ND          .3233   .0910                                        22.5         ND          ND      .0795                                        23.0         ND          ND      .3910                                        23.5         ND          ND      .2917                                        24.0         ND          ND      .2415                                        Anticoagulant                                                                              .3330       ND      .1960                                        Activity of                                                                   Plasma of                                                                     Sacrificed Rat                                                                ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Rat No.          155        160    161                                        ______________________________________                                        Tritiated Heparin Complex                                                                      74.30      23.87  20.53                                      Transported in Lymph, %                                                       Tritiated Heparin Complex                                                                      ND          4.94  11.87                                      Remaining in GI Tract %                                                       ______________________________________                                    

COMPARATIVE EXAMPLE 1 Oral Administration of Heparin

Commericially available heparin was orally administered to a group ofrats. Six rats were employed in this study. They had an average weightof 415.5 grams with a standard deviation of 47.1 grams. The heparinorally administered dosage averaged 3992 units per kilogram with astandard deviation of 373 units per kg with a standard deviation of 2.39mg/kg. The anticoagulant activity, measured by the APTT method, wasmeasured as a function of time and is summarized in Table 3 below.

                  TABLE 3                                                         ______________________________________                                        Time After Oral Heparin                                                                        Anticoagulant Activity,                                      Administration, hr                                                                             unit/ml                                                      ______________________________________                                        0                0                                                            .25              .03 ± .04                                                 .50              .03 ± .05                                                 .75              .05 ± .06                                                 1.00             .03 ± .05                                                 1.50             .03 ± .06                                                 2.0              .03 ± .06                                                 3.0              .02 ± .04                                                 4.0              0                                                            5.0              .03 ± .04                                                 6.0              0                                                            7.0              0                                                            ______________________________________                                    

The above embodiments and examples are given to illustrate the scope andspirit of the present invention. These embodiments and examples willmake apparent, to those skilled in the art, other embodiments andexamples. These other embodiments and examples are within thecontemplation of the instant invention. Therefore, the invention shouldbe limited only by the amended claims.

What is claimed is:
 1. An enterally effective, biologically activepeptide or protein composition, comprising:a sandwich complex comprisinga hydrophobic core complex of a biologically active peptide or proteinwith an alkyl or alkenyl sulfate having 6-24 carbon atoms and 0-3 doublebonds, said core complex forming an electrostatic complex with aquaternary ammonium ion selected from the group consisting of: ##STR27##where R¹ and R² are the same or different and are alkyl or hydroxysubstituted alkyl containing 1 to 6 carbon atoms; and R, R' and R" arethe same or different and are saturated or unsaturated aliphaticscontaining at least 10 carbon atoms; ##STR28## where R¹, R² and R" havethe meanings given above; ##STR29## where R³ is independently alkyl orhydroxy substituted alkyl containing at least 10 carbon atoms; and R¹,R², R and R' have the meanings given above; and ##STR30## where R¹, R²and R³ have the meanings given above.
 2. The composition of claim 1,wherein said sulfate is an alkyl sulfate.
 3. The composition of claim 2,wherein said alkyl sulfate contains from 10 to 14 carbon atoms.
 4. Thecomposition of claim 2, wherein said alkyl sulfate is dodecyl sulfate.5. The composition of claim 1, wherein the ratio of said alkyl sulfateto said quaternary ammonium is a 1:1 molar ratio.
 6. The composition ofclaim 1, wherein said peptide or protein is present in a weight ratio ofabout 1:0.4 with respect to said alkyl or alkenyl sulfate.
 7. Thecomposition of claim 1, wherein said peptide or protein is present in aweight ratio of about 1:1.4 with respect to said alkyl or alkenylsulfate.
 8. The composition of claim 2, wherein said biologically activepeptide or protein is a peptide hormone.
 9. The composition of claim 8,wherein said hormone has a molecular weight of less than
 7000. 10. Thecomposition of claim 2, wherein said peptide or protein is insulin. 11.The composition of claim 10, wherein said alkyl sulfate contains 10, 12or 14 carbon atoms.
 12. The composition of claim 11, wherein saidalkylsulfate is dodecyl sulfate.
 13. The composition of claim 12,wherein the weight ratio of insulin to dodecyl sulfate is about 1:1.4.14. The composition of claim 10, wherein the weight ratio of insulin tododecyl sulfate is about 1:0.4.
 15. The composition of claim 13, whereinsaid quarternary ammonium ion is present in a 1:1 molar ratio to saidalkyl sulfate.
 16. A method of producing an enterally effective,biologically active peptide or protein composition, comprising the stepsof:dissolving a biologically active peptide or protein in an aqueoussolvent to form a solution; adding an alkyl or alkenyl sulfate having6-24 carbon atoms and 0-3 double bonds to said solution to form ahydrophobic core complex; adding a quaternary ammonium ion to thesolution of said core complex, wherein said ammonium ion is selectedfrom the group consisting of ##STR31## where R¹ and R² are the same ordifferent and are alkyl or hydroxy substituted alkyl containing 1 to 6carbon atoms; and R, R' and R" are the same or different and aresaturated or unsaturated aliphatics containing at least 10 carbon atoms;##STR32## where R¹, R² and R" have the meanings given above; ##STR33##where R³ is independently alkyl or hydroxy substituted alkyl containingat least 10 carbon atoms; and R¹, R², R and R' have the meanings givenabove; and ##STR34## where R¹, R² and R³ have the meanings given abovethereby forming a sandwich complex comprising said hydrophobic corecomplex in an electrostatic complex with said ammonium ion.
 17. Themethod of claim 16, wherein said sulfate is an alkyl sulfate.
 18. Themethod of claim 17 wherein said sulfate is dodecyl sulfate.
 19. Themethod of claim 16, wherein said peptide or protein is a peptidehormone.
 20. The method of claim 19, wherein said hormone is insulin.21. The method of claim 16, wherein said sulfate is dodecyl sulfate andsaid peptide or protein is insulin.
 22. A pharmaceutical composition,comprising: the sandwich complex of claim 1 and a pharmaceuticallyacceptable carrier.
 23. The composition of claim 22, wherein saidcarrier is water or an aqueous solution of a non-toxic salt.
 24. Thecomposition of claim 22, wherein said carrier is a dilute aqueoussolution of a mineral acid having a pH of less than
 4. 25. Thecomposition of claim 24, wherein said carrier is 0.005M phosphoric acid.26. The composition of claim 22, wherein said complex is lyophilized andsaid carrier is a solid.
 27. A method of treating diabetes comprisingenterally administering to a diabetic human or animal an amount of thesandwich complex of claim 10 sufficient to produce a hypoglycemiceffect.
 28. The method of claim 27, wherein said administering is orallyadministering.